Device for selective destruction of cells

ABSTRACT

Apparatus for selective destruction or inactivation of cells is disclosed wherein cells are arrayed in a plane for successive illumination, by optical X-Y scanning devices, with a first low power red light beam to produce in particular cells certain radiations responsive to the illumination. These response radiations are detected and used to enable a second higher powered light beam directed through substantially the same optical scanning paths, to destroy or inactivate the cells producing the response radiations. The illumination, response, detection, and high power radiation steps are accomplished by devices acting in times short as compared to the X-Y scanning devices so as to make the accurate treatment of each of large numbers of cells highly effective. Alternate embodiments disclose the use of a signal laser light source to selectively provide both the low and high powered light beams, various X-Y scanning arrangements, and various devices for correcting the spherical aberration of the scanned beams in variously positioned image planes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to apparatus for the selectivedestruction of cells, and more particularly to a system of the typewherein cells arrayed in a plane are successively illuminated with a lowpowered light beam and examined for particular radiation resulting fromthis illumination to selectively enable a high powered beam toaccomplish the destruction of particular cells.

2. Description of the Prior Art

From the German patent document DE-OS 53 31 969, a flow cytometry deviceis known for detection of interesting biological particles in a sampleof unknown particles, in which a beam from a light source is directedonto the particles, and light related data is detected by a measuringdevice if the light beam strikes a particle. In this case the measuringdevice detects, inter alia, the fluorescence emitted by certainparticles.

From the German patent document DE-PS 33 31 017, a process is disclosedfor distinguishing various types of cell subpopulations, in whichantibody proteins are labeled with fluorochromes; the labeled antibodyproteins are combined with a sample of cells, in which specificreceptors for the labeled antibody proteins are suspected; thefluorochromes are stimulated by suitable techniques and the cells areanalyzed for classification on the basis of emitted fluorescence.

SUMMARY OF THE INVENTION

It is a primary object of the present invention to provide apparatus inwhich cells, especially blood cells, are optically characterized andselectively destroyed with light beams to achieve a therapy or to studythe interaction of blood cells.

A substantial advantage of the present invention is that it now becomespossible, depending on specific syndromes or problems, to selectivelydestroy cells, especially blood cells negatively influencing or causingthe syndrome, and in this way to achieve an improvement in or assist inthe healing of the pathological process. Preferably this can take placelike a blood washing, by the blood being branched off from the patient'sbody and fed to the device according to the present invention. Withinthe device, the blood cells causing or influencing the disease, inwhich, for example, certain lymphocyte populations are involved, areselectively destroyed, and the blood is then reintroduced into the bloodcirculation of the patient. By destruction of these blood cells thesymptoms of the disease tend to be improved, or healing is assisted.Especially, it is possible that with the help of the device according tothe present invention diseases such as leukemias, rejection phenomena intransplants, AIDS and autoimmune diseases can also be effectivelytreated.

A substantial advantage of the device according to the invention is thatsyndromes, especially the above mentioned syndromes, can be treatedwithout the patient's health being endangered by side effects, as in thecase, for example, in chemotherapy.

In an advantageous way the invention makes it possible for differentsyndromes to be treated with one and the same device, and only dependingon the disease to be treated, the use of characteristic properties ofthe individual blood cells or the use of different antibody RNA samplesor other labeling materials is necessary.

This device can advantageously be used both for treatment of staticblood and flowing blood. It is also possible with this device to treatcell suspensions on a large scale.

The invention is also advantageously used for purification ofbiotechnologically usable cell suspensions. In addition, this device canalso advantageously be used in the field of chromosome analyticseparation of cells.

Another advantage is that the device according to the invention cansubstantially be produced from components available on the market.

In a preferred embodiment, and by means of alternate embodiments, thepresent invention discloses apparatus for providing the above advantagesby carrying out the following steps. In this illustrative case, blood isused as "cell suspension" to assist in the explanation.

(1) A patient's blood is branched from the circulation by,illustratively, an extracorporeal circulation.

(2) An individual cell group, e.g., erythrocytes, leukocytes,lymphocytes or thrombocytes, is taken from the blood by a cell selectionunit, since it can contain relevant cells for certain syndromes. Therest of the blood cells are fed back into the blood circulation by acell feed device.

(3) The removed group contains cells that are relevant and irrelevantfor the syndrome, cells which cannot be distinguished from one anothermorphologically and physically. For this reason, selection of therelevant blood cells takes place by use of fluorescent antibodies, DNAsamples, RNA samples or other labeling means directly or indirectly.

(4) All blood cells of the blood cell group are brought by a scanningdevice into the focal or image plane of an optical system and aresuccessively illuminated with a light beam with a power harmless to theblood cells. Because of their labeling, the relevant blood cells emit afluorescence.

(5) The light emitted by the labeled blood cells is detected by a sensorwithin a detector unit.

(6) By radiation of a light beam within an active unit, with a higherpower which is sufficient for destruction of the blood cells, into theoptical system, labeled or relevant cells, which are responsible for thesyndrome, can be destroyed. In this case, the light beam with the higherpower is activated in that the fluorescence of the labeled or relevantcells, after their stimulation by the light beam with low power, isdetected for generation of an activation signal.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional objects and advantages of the invention will become apparentto those skilled in the art as the description proceeds with referenceto the accompanying drawings wherein:

FIG. 1 is a simplified block diagram outlining the overall sequence ofoperation of the system according to the present invention;

FIG. 2 is a detailed block diagram of a preferred embodiment of thepresent invention;

FIG. 3 shows a simplified side view of a belt conveyor employed in anembodiment of the present invention;

FIG. 4 shows a simplified cross-sectional view of an alternateembodiment image plane arrangement;

FIG. 5 diagrammatically shows a first spherical aberration correctiontechnique for use in the present invention; and

FIG. 6 diagrammatically shows an alternate spherical aberrationtechnique.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Operation of the present invention is first explained in general termsin connection with the simplified block diagram of FIG. 1. In FIG. 1,the device according to the present invention is shown as consistingsubstantially of an analysis unit 1, a scanning device 2, an active unit3, a cell feed device 4, and a detector unit 7. In this arrangement, thecell feed device 4 can suitably be connected upstream from a cellpreselector 5.

The analysis unit 1 produces at least one light beam 6 with a powerharmless to cells, especially for blood cells. This light beam 6 isprojected by the scanning device 2 onto an image plane of an opticalsystem and is advantageously scanned there in two directions (X and Y ofa Cartesian coordinate system) if the image plane itself is stationary;or is scanned only in one direction of such coordinate system, while theimage plane is moved in the other direction. Alternatively, it is alsopossible for the light beam 6 to be projected only onto the image planeby the scanning device 2, and only the image plane is moved in a presetdirection, so that no surface, as in the two preceding scanning cases,but only a line is scanned by the light beam 6.

The detector unit 7 detects the fluorescence emitted by individual cellssuccessively stimulated by light beam 6 in the image plane and/or thelight radiation reflected on these cells and/or the radiation scatteredon these cells and/or the radiation emitted by the cells. In FIG. 1 saidemitted fluorescence and other response light beams are identified bythe path 8. The detector unit 7 generates at least one detector signal9, which corresponds to the detected emission and/or certain scatteredor reflected radiation 8.

At least one detector signal 9 is applied to control devices withinactive unit 3, and an activation signal is generated which activates alight source contained in active section 3. This light source producespower light beam 10, whose power is so high that it is sufficient fordestroying or inactivating cells. This power light beam 10 is preferablyfed to the optical system of scanning device 2, so that it is projectedon the same point of the image plane as was light beam 6. This meansthat with stimulation of a cell by light beam 6 and with reception of apredetermined fluorescence emitted by this cell, or of a predeterminedradiation reflected or scattered on this cell, the activation signal andpower light beam 10 are produced; since in the case of the cell alabeled relevant cell responsible for a syndrome is involved. This cellis then destroyed by the power light beam 10. It is also possible forthe analysis steps (projection of light beam 6) and the steps of activeintervention (power light beam 10) to be performed separately in spaceand time.

The cell feed device 4 feeds the cells to be examined or treated into animage plane within scanning device 2. In this case the fed cells, ifblood cells are involved, can be taken via pathway 16 from a patient'sblood circulation 11 as shown diagrammatically, and can be fed back inthis blood circulation after therapy or examination has been performed.In the case shown, cell preselector 5 can be used. It draws out from theblood fed to it, illustratively by a centrifugal operation, anindividual cell group, which may contain the cells relevant for thesyndrome to be treated. In this case, only the removed relevant bloodcells are brought by the cell feed device 4 into the image plane of thescanning device 2. The remaining blood cells of the blood branched offfrom the blood circulation 11 are fed back to it immediately after cellpreselector 5 via pathway 12, while the treated blood cells are fed backinto the blood circulation 11 from the image plane via pathway 13. Bymeans of this preselection, not all the blood cells of the blood supplybut only the blood cells of an individual cell group, in whichpreferably the cells responsible for the syndrome to be treated arecontained, must be scanned. Consequently, the entire scanning procedurecan be substantially accelerated.

Preferred embodiments of this invention are now explained in more detailin connection with FIG. 2. Elements shown in FIG. 2 which were explainedin connection with FIG. 1 are correspondingly designated.

In the analysis unit 1, a laser source is preferably used as a lightsource 51, since in this case a particularly sharp and small focal spotcan be produced in an image plane 21 of the scanning device 2. Thisleads, for example, to the production of well defined fluorescences ofthe cells successively scanned in the image plane 21.

An argon laser is particularly well suited as laser light source 51,whose stimulation frequency corresponds, illustratively, to 488 nm.Antibodies labeled with fluorescein isothiocyanate, if they arestimulated with 488 nm of the corresponding stimulation frequency, thenemit a fluorescence at 550 nm. With laser sources, focal spots caneasily be produced whose diameter is about 10 microns or less.

Instead of a single light source 51, several light sources can beprovided, e.g., two light sources 51 and 52, which produce light beams61 and 62, respectively, whose power is harmless to the cells. When twolight sources 51 and 52 are used, the cells can be stimulated in theimage plane 21 at the same time with different frequencies so that, forexample, two fluorescences with different wavelengths are produced. Thiscan be advantageous for special problems or therapy. Moreover, this canbe especially important for generation of trouble-free detector signals9, since, by subtraction of the two signals detected by correspondingsensors of detector unit 7, in which fluorescence signals,illustratively, are involved, background signals can be eliminated.

The two light beams 61 and 62 are brought together into one beam and arepicked up together by the optical system of scanning device 2. Theoptical system of scanning device 2 projects a focal spot of lightsource 51, or light sources 51 and 52, onto the image plane 21. Scanningdevice 2 includes two deflection mirrors 57 and 58, known in the art,one of which is shifted by a drive device for the deflection in an Xdirection, and the other is shifted by another drive device fordeflection in a Y direction while oscillating. Instead of deflectionmirrors 57 and 58, so-called electrooptical deflectors can alternativelybe provided, which also are known in the art. In addition, deflection inthe X and Y directions can be achieved in a way known in the art byother rotating optical means.

By using only one deflection mirror or only one electroopticaldeflector, the deflection of light beam 61 or light beams 61 and 62 canoccur in only one direction (for example, in the X direction). In thisarrangement there is the possibility that image plane 21 itself may bemoved in the other direction (for example, in the Y direction). This cansuitably take place by means of a belt conveyor 22. In this arrangement,the image plane corresponds to a part of the surface of belt 23 and thedeflection of light beam 61, or light beams 61 and 62, preferably takesplace perpendicular to the direction of movement of the belt 23. Byproviding a transparent belt 23, a sensor device 71 of detector unit 7,which is explained below in greater detail, can be provided on the sideof belt 23 opposite image plane 21. In case of use of a belt conveyor22, the blood from a suitable storage 24 is properly put on the surfaceof belt 23 together with nutrient medium that keeps the blood cellsalive; viewed in the direction of movement of belt 23, before the placeon which light beam 61, or beams 61 and 62, is scanned.

Referring now to FIG. 3, an illustrative belt conveyor device isdescribed in which the blood is put on one end of belt 23, and in whichthe blood cells during the movement of belt 23 to a scanning site 25 areso settled that they are placed exactly at scanning site 25 in the imageplane on the belt surface. To achieve this, the rate of movement of belt23 is set as a function of the settling process. To prevent lateralflowing off of the blood from belt 23, the belt includes side walls 26,so that the belt surface corresponds to the bottom of a formed,approximately U-shaped duct.

In a preferred alternate embodiment of the present invention as shown inFIG. 4, the image plane is formed by the surface of a transparent plate27 and the scanning device 2 is alternately configured so that lightbeam 61, or beams 61 and 62, impact from below onto the plate 27. Thenthe blood which may be contained in a transparent plastic bag 28, or thelike, or the blood cells selected by cell preselector 5 contained in thebag 28 can be placed on the opposite surface of plate 27. In thisalternate embodiment, light beam 61, or beams 61 and 62, are focused onthe plane of the cells resting on the bottom of bag 28. In this case, itis advantageous that the power light beam 10 focused on the individualcells with power leading to the destruction of relevant cells in thearea, in going through the wall of bag 28, is not yet focused, so thatthe power density is so low that the bag is not destroyed or damaged.

Light beam 61, or beams 61 and 62, can also be focused on a stationarypoint of image plane 21, and the blood or blood cells flow through theareas of the point, as is known in connection with the so-called flowchambers.

Referring now back to FIG. 2, it is seen that the detector unit 7includes sensors 71 and 72 corresponding, illustratively, to the pair oflight sources 61 and 62. These sensors receive the fluorescence emittedby the cells 73 and 74, or radiation reflected or scattered on thecells, and generate detector signals 75 and 76 upon receiving a certainfluorescence or certain radiation. Preferably the fluorescence emittedby the cells or the radiation reflected on the cells is fed back by theoptical system of scanning device 2, and the fluorescence signals orreflected light signals 73 and 74 are suitably supplied by beam dividers77 and 78 from the beam path of the optical system to sensors 71 and 72.But it is also possible to provide sensors 71 and 72 of detector unit 7at the side of image plane 21. This is especially suitable in the caseof analysis of the radiation scattered on the cells. Additionally, glassfibers can also be used for guiding the various light beams, as is knownin the art.

Detector signal 75, or detector signals 75 and 76, are fed to a controldevice 31 within the active unit 3, in which a central processing unit(CPU) is involved, to generate an activation signal from the detectorsignal(s). Preferably, photomultiplier tubes are used as sensors 71 and72, upstream from which one or more filters 79 are installed. Thesefilter(s) pass only one wavelength, which corresponds precisely to theemitted fluorescence or other radiation of interest.

The activation signal from the control device 31 is fed to a lightsource 32 of active unit 3, which produces the power light beam 10 withthe power and wavelength which are suitable for destruction orinactivation of the cells. For this light source 32, a laser source,especially an argon laser, is preferably involved. It is also possibleto produce UV light by this light source 32. Power light beam 10 oflight source 32 is supplied by a beam divider 33 into the beam path ofthe optical system of scanning device 2.

In an alternate embodiment, instead of the aforementioned light source51 with low power, and the light source 32 with high power, a singlelight source 15 can also be provided. This single light source 15normally produces a light beam 14 with low power which is harmless tothe cells, and the power of this light beam 14 in the presence of theactivation signal from the control device 31 can momentarily be soincreased that it is sufficient for destruction or inactivation ofcells. After a preset period, which may be from about 30 to 40 nsec, theenergy of light beam 14 in each case is again reduced to the low energylevel and the scanning process is continued. For the light source 15, aknown laser device of the so-called acoustooptic cavity dumping type maybe used; which in the presence of the activation signal can produce in arise time of 7 nsec, a high-energy pulse of 36 nsec.

Possible solutions are explained below by which even with greatdeflections of the focal spot of light beam 61, or beams 61, 62 and 10in the image plane 21, a correction of the spherical aberration isachieved. A novel idea for solving this problem consists in the use of alens rotating on a disc, which is synchronized with the movement ofdeflection mirrors 57 and 58 or with the control of the alternateelectrooptical deflectors. FIG. 5 shows such an arrangement. A disk 81eccentrically carrying a lens 83 is so rotated, and deflection mirrors57 and 58 (or the electrooptical deflectors) were so driven orcontrolled, that it is always assured that light beams 61, and 62 or 10,all guided by the common optical system of scanning device 2, alwaysaccurately follow the rotation of lens 83 during a preset period. Sincethe lens 83 describes a circular path, a deflection in the X-Y directionis necessary. Then in the image plane 21 a circular deflection line 84of the focal spot or spots occurs. Scanning in the other direction (Ydirection) is achieved by the image plane 21 being shifted in thedirection of arrow 80 relative to deflection line 84. This occurs, forexample, with belt conveyor 22 already mentioned.

Especially preferred in this arrangement is an embodiment in which twolenses 83 and 83' are placed equally distant from the center on adiameter of rotating disk 81 so that with a suitable synchronization ofdisk 81 and of deflection mirrors 57 and 58 (or of the electroopticaldeflectors) a lens 83 or 83' is always rotated exactly into beam 61 and62 or 10 returned to a beginning position A to follow the beam to an endposition E. Broken line 86 shows the return of the beam from endposition E to beginning position A.

It is also possible, instead of rotating disk 81 with lens 83, or lenses83 and 83', to provide a lens 87 on an oscillating plate 88, which ismoved back and forth in a straight line 89, as shown in FIG. 6. In thisarrangement, only one deflection mirror or one electrooptical deflectoris necessary, which is synchronized with the back and forth movement ofplate 88 so that the light beam always goes through lens 87. Straightdeflection line 80 then is produced in image plane 21. The advantage ofusing rotating disk 81 and oscillating plate 88 reside in the fact thatlenses of high aperture can be used and thus a great sensitivity can beachieved.

It is further possible for correcting the above described sphericalaberration, to place a dynamic lens 91 (as shown in FIG. 2) known in theart, in the beam path such a lens can be shifted in the beam directionas a function of a signal that shows that the focal spot is not in theimage plane 21. In this way, it is possible for dynamic lens 91 to beautomatically guided so that the focal spot is always exactly in imageplane 21. Such dynamic lenses are known, for example, in connection withCD phonographs.

Additionally, in lieu of rotating disk 81, in the beam path ahead ofimage plane 21 a so-called flat field lens (F lens) can be provided,whose characteristics in the X deflection direction and/or Y deflectiondirection change so that it is always assured that the deflected focalspot is always in image plane 21. Such a flat field lens is showndiagrammatically as lens 92 in FIG. 2.

Instead of the described laser source producing the power light beam 10,sources producing shock waves can also be used. Moreover, certain cellscan be labeled with photolabile substances, which release intracellularpoisons if light of suitable wavelength and energy strikes them as beam10.

In the detection and evaluation of the light reflected or scattered onthe blood cells an image or special optically detectable features can berecorded and these can be compared with a previously stored image orpreviously stored optical features which characterize certain blood cellor cells to be destroyed. In the case of agreement of the detected andstored data, destruction of the blood cells present at the moment can betriggered by generation of the activation signal.

In an illustrative contemporary usage of the present invention, apossible AIDS therapy is described. T4 lymphocytes infected withRTLV-III viruses incorporate (after activation with Interlencin I andantigen) virus protein in the T4 cell membrane. Therefore, it seemspossible selectively to label the infected cells with fluorescencelabeled antibodies relative to the virus proteins. Illustratively, anassumption is made that 5 liters of blood, which contains 8000leukocytes per microliter, is treated. This means that 5×10⁶×8000=4×10¹⁰ leukocytes are in 5 liters of blood. It is further assumedthat 50%, i.e., 2×10¹⁰, are T-lymphocytes. The blood cells have adiameter of about 10 microns. This means that the focal spot of laserbeam 10 used for selective cell destruction is to have a diameter ofabout 10 microns. Such focal spot sizes are easily attainable.

If the assumption is that 10¹⁰ cells are closely pressed against oneanother, an area of about 10¹⁰ ×10×10⁻⁶ ×10×10⁻⁶ =1 m² is covered. Eachcell is to be selectively illuminated within 10,000 sec., i.e., about2.7 hours. This corresponds to a frequency of 10¹⁰ cells/10⁴ equal to10⁶ hertz. For the scanning of a blood cell about 1 microsecond isavailable which is technically possible. During the scanning, the cellscan be cooled, for example to 4° C., by a moderating device, not shown.Thus, e.g., the fluorescence is stabilized.

We claim:
 1. A system for selective destructive or inactivation ofcells, wherein planar arrays of cells are successively illuminated witha lower powered light beam produced by at least one light source and theradiation of particular cells responsive to said illumination enables ahigher powered beam routed via a substantially common optical path toaccomplish said destruction or inactivation in a near simultaneousmanner, said system comprising:(a) means defining an image plane forgroups of cells; (b) at least one laser light source for producing atleast a first light beam to illuminate groups of cells positioned insaid image plane; (c) scanning means for directing said at least firstlight beam within said image plane to successively illuminate saidgroups of cells in the image plane; (d) at least one optical detectorfor sensing predetermined radiations occurring from a particular cell ofsaid groups of cells at a particular point of said scanning means inresponse to said illumination, and for producing at least one detectorsignal responsive to said sensed predetermined radiations; (e) controlmeans operating in response to said detector signal to enable a secondlight beam to be directed through said scanning means to said particularpoint, wherein the power level of said second light beam is sufficientto destroy or inactivate said particular cell; (f) said control meanseffecting activation of said second light beam before said scanningmeans has moved appreciably from said particular point; (g) said firstlight beam and said second light beam being formed by a single lightsource; and (h) wherein said control means permits a single light beamto be switched from a low power harmless to cells to a higher powerharmful to cells upon detector signal production wherein said scanningmeans comprises mirrors for deflecting said light beams in X-Ydirections of a Cartesian coordinate system.
 2. The system according toclaim 1 wherein said single light source laser is an argon laser.
 3. Thesystem according to claim 1 wherein one of said deflection mirrorsdeflects said light beams in a first of said X-Y directions of saidimage plane and said image plane is movable in a second of saiddirections.
 4. The system according to claim 3 wherein said scanningmeans further comprise a dynamic lens positioned in front of saidmirrors for correction of spherical aberration in said beams at saidimage plane.
 5. The system according to claim 4 wherein said scanningmeans further comprise a flat field lens for correction of the sphericalaberration between said image plane and said mirrors.
 6. The systemaccording to claim 1 wherein said image plane corresponds to an insidebottom surface of a vessel in which the cells are placed.
 7. The systemaccording to claim 1 wherein said image plane is formed by at least onepartial area of the surface of a belt of a conveyor.
 8. The systemaccording to claim 7 wherein said belt consists of a transparentmaterial, and said optical detector is placed on the side of the beltopposite to said image plane.
 9. The system according to claim 7 whereinsaid belt includes outwardly projecting side walls on its sides so thata duct is formed for receiving the cells.
 10. The system according toclaim 1 wherein a cell feed device feeds blood cells taken from apatient to said image plane.
 11. The system according to claim 10,wherein upstream from the cell feed device there is connected to thecell feed device a preselector separating blood cells and preselectedcells said patient's blood circulation.
 12. The system according toclaim 1 wherein said scanning means further comprises a rotatable lenseccentrically fastened to a rotatable disk which may be moved insynchronization with said deflection mirrors.
 13. The system accordingto claim 1 wherein said scanning means includes in front of said imageplane an oscillation plate with a lens whereby said oscillation platecan be moved in one direction while oscillating in synchronization withan oscillation of one of said deflection mirrors.